The hemolymph was collected
from G. mellonella larvae, placed in freezer for 10
min to stop the larval movement, followed by decapitation.
Hemolymph was collected from 20 fifth instar larvae by incising
the abdominal proleg and squeezing the hemolymph
into 15-ml falcon tubes. The tubes were centrifuged at 10,000
× g for 5 min, and the hemolymph was collected in fresh
tubes. PO inhibition activity was assayed in 96-well plates
with 100 μl 50 mM phosphate buffered saline (PBS) containing
2 mM L-DOPA added to 20 μl compound 1 and/or
benzaldehyde. PO activity was monitored by measuring absorbance
at 495 nm using a plate reader (Dynatech MR5000,
El Paso, TX, USA) over a period of 90 min. It should be noted
that this approach predominantly detects dopachrome and/or
dopaminechrome rather than melanin itself.